http://informahealthcare.com/dct ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, 2014; 37(3): 303–310 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2013.851690
RESEARCH ARTICLE
A subchronic (180-day) oral toxicity study of ethyl tertiary-butyl ether, a bioethanol, in rats Katsumi Miyata, Takayuki Koga, Sunao Aso, Satsuki Hoshuyama, Syozo Ajimi, and Kotaro Furukawa CERI Hita, Chemicals Evaluation and Research Institute, Hita, Japan
Abstract
Keywords
A subchronic (180-day) toxicity study was conducted to evaluate the effects of ethyl tertiarybutyl ether (ETBE), a biomass fuel, in male and female rats. ETBE was administered at dose levels of 0, 5, 25, 100 and 400 mg/kg/body weight (b.w.)/day by gavage. No treatment-related adverse effects were observed at 5, 25 or 100 mg/kg. Centrilobular hypertrophy of hepatocytes was observed in males and females and their relative liver weights were increased, suggesting enhanced metabolic activity. From these results, we concluded that the no observed adverse effect level of ETBE was 100 mg/kg b.w./day under the conditions tested.
Ethyl tertiary butyl ether (ETBE), oral, rat, subchronic toxicity
Introduction Ethyl tertiary-butyl ether (ETBE) is industrially produced from ethanol and isobutylene and commonly used as an oxygenated additive of gasoline. ETBE offers equal or greater air-quality benefits compared to ethanol while being technically and logistically less challenging. ETBE does not induce evaporation of gasoline, which is one of the causes of smog, and does not absorb moisture from the atmosphere (EFOA, 2006; Fujii et al., 2010). The Japanese government has developed the Kyoto Protocol Achievement Plan, in which biomass fuels are planned to be introduced at 500 000 kL of crude oil equivalent per year for transportation purposes. To achieve this goal, the Japanese oil industry decided to introduce 840 000 kL of bioETBE as up to 7% ETBE-blended gasoline for automobile fuel from 2010. The Japan Petroleum Energy Center (JPEC) and the Ministry of Economy, Trade and Industry, Japan (METI), have been leading the project to assess the health risks of ETBE before the registration of ETBE pursuant to Japan’s chemical substance control law (1973). A series of analytic and toxicological studies demonstrated that ETBE is a persistent chemical in terms of biodegradability, but ETBE is not very bioaccumulative and is neither mutagenic nor ecotoxic. However, ETBE induced an increase of liver weight and centrilobular hepatocyte hypertrophy in rats after a 28-day repeated oral toxicity study (Miyata et al., 2004). Based on these results, ETBE was designated as a type II monitoring chemical substance according to the Japanese act, which Address for correspondence: Katsumi Miyata, Hita Laboratory, Chemicals Evaluation and Research Institute, 822 Ishii-machi 3chome, Hita 877-0061, Oita, Japan. Fax: +81-973-23-9800. E-mail: [email protected]
History Received 11 April 2013 Revised 26 August 2013 Accepted 13 September 2013 Published online 19 November 2013
means that the substance is not likely to undergo chemical transformation through natural processes. Systemic inhalation toxicity of ETBE has been well characterized in subchronic (90-day) studies using rats and mice. ETBE caused transient ataxia in Fischer 344 rats at a dose level of 5000 ppm (Dorman et al., 1997). Transient ataxia was also found in another subchronic inhalation study (Medinsky et al., 1999), in which both Fischer 344 rats and CD-1 mice exhibited the abnormality at 5000 ppm. Other major toxic findings observed in rats of the latter study were increased growth rate and slightly increased heart weights in females, degenerations of testes in males, and increased kidney and liver weights in both sexes. Mice have shown ataxia and liver abnormalities, such as increased weight, increased rate of cell turnover and/or centrilobular hypertrophy in males and females (Medinsky et al., 1999). Based on these results, the no observed adverse effect level (NOAEL) of ETBE for systemic toxicity was estimated to be 500 ppm in both rats and mice. However, long-term oral toxicity studies using rats and mice have hitherto not been conducted. The present study aimed at examining the potential longterm systemic toxicity of ETBE administered by the oral route for precise risk assessment, following Organization for Economic Cooperation and Development (OECD) test guideline 452 (OECD, 1981) chronic toxicity studies, in principle, with an exposure period of 180 days.
Methods Materials ETBE (CAS no.: 637-92-3) was supplied by Tokyo Chemical Industry Co. Ltd. (lot no. R74EE, 99% in purity; Tokyo,
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Japan). Stability of ETBE was verified by infrared spectroscopy before and after the dosing period, and purity was confirmed by gas chromatography analysis. Animals Specific pathogen-free Crl:CD (Sprague-Dawley) rats were used in this study. Eighty animals of each sex were purchased from Hino Breeding Center, Charles River Laboratories Japan, Inc. (Yokohama, Japan) at 4 weeks of age. After a 6-day quarantine and acclimatization period, 75 healthy animals of both sexes were selected. Animals were weighed and assigned to five groups in such a way as to equalize group means and standard deviations (SDs) of body weights. Each group consisted of 15 males and 15 females. Animals were 5 weeks of age at start of dosing and maintained in a barrier-sustained animal room with controlled temperature (21–25 C), humidity (40–70%), ventilation (10–15 times per hour) and lighting cycle (12 hours of artificial light [7:00 a.m. to 7:00 p.m.] and 12 hours of darkness) and were given an autoclaved pellet diet (MF; Oriental Yeast Co., Ltd., Tokyo, Japan) and local tap water ad libitum. They were housed individually in metallic brackettype cages with wire mesh floors throughout the study. Test substance administration ETBE was dissolved in olive oil and orally administered to animals with a stomach tube once-daily at approximately the same time in the morning. The oral route is one of the most probable routes of human exposure to ETBE, because leakage of ETBE from a gasoline tank may contaminate underground water. Dosing volume was 5 mL/kg body weight (b.w.), and the volume for each animal was calculated based on the most recent body weight. Dosing solutions were analyzed for the concentration of ETBE and were used within 7 days after preparation. Stability of ETBE in dosing solutions during the dosing period was confirmed by chemical analysis (data not shown). Dose levels were selected based on the results of a 28-day repeated-dose oral toxicity study in rats (Miyata et al., 2004), in which Crl:CD (Sprague-Dawley) rats, 5/sex/group, received ETBE orally at dose levels of 0, 15, 25, 50, 100, 150, 400 and 1000 mg/kg/day for 28 days. This short-term study revealed decreased spontaneous movement at 100 mg/kg and above, along with increased weights of the liver at 400 mg/kg. Therefore, a dose level of 400 mg/kg b.w./day was selected for the highest dose for the definitive study, and the lower dose levels were set at 100, 25 and 5 mg/kg b.w./day. Control animals received the vehicle only in the same manner as for the test-substance-treated groups. Study design This study was conducted in compliance with the OECD Chronic Toxicity Study (OECD, 1981) guideline for test chemicals in principle, with some modifications, and complied with the principles for Good Laboratory Practice (OECD no. 26, November 1997). The study also followed the Act on Welfare and Management of Animals of the Ministry of the Environment, Japan (Act No. 105, October 1, 1973), and Basic Policies for the Conduct of Animal Experiments in
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Research Institutions under the Jurisdiction of the Ministry of Health, Labor and Welfare, Japan (June 1, 2006). Any clinical signs were recorded daily, and individual animal weights and food consumption were measured once-weekly for the first 13 weeks of administration and once-monthly thereafter. Detailed clinical observations were performed once-monthly. During the last month of administration, reflex, grip strength and locomotor activity were assessed. Hematology and serum biochemistry Blood samples were obtained on the next day of the last treatment. Animals were fasted overnight, and blood samples were collected from the abdominal aorta of each 10 animals of a group under deep ether anesthesia. Hematological parameters were assessed on red blood cell (RBC) count, white blood cell (WBC) count, hemoglobin (Hb) concentration, hematocrit (Ht) value, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, reticulocyte count, prothrombin time (PT), activated partial thromboplastin time (APTT) and differential leukocyte count. Clinical chemistry parameters determined in serum samples were glutamic-oxaloacetic transaminase (AST), glutamicpyruvic transaminase (ALT), alkaline phosphatase (ALP), cholinesterase (Che), gamma-glutamyl transpeptidase (GGT), total cholesterol (T-Cho), triglyceride (TG), glucose, total protein, albumin, blood urea nitrogen (BUN), creatinine, total bilirubin (T-Bil), calcium, inorganic phosphorus (IP), sodium, potassium and chlorine as well as the calculated albuminglobulin (A/G) ratio. Urinary parameters were assessed on urine volume, color, turbidity, specific gravity, pH, protein, glucose, occult blood and urinary sediment. Pathology After blood samples were collected, all animals, including 10 animals of the clinical examination, were sacrificed humanely and underwent complete necropsy, including external examination. Tissues and organs collected from males and females, as appropriate, consisted of gross lesions, the trachea, lungs, submandibular and sublingual glands, esophagus, stomach, intestine (duodenum, jejunum, ileum, cecum, colon and rectum), pancreas, liver, heart, aorta, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicles, ovaries, uterus, vagina, brain (cerebrum, cerebellum and medulla/ pons), spinal cord (cervical, thoracic and lumbar), sciatic nerve, bone marrow (sternum and femur), axillar and mesenteric lymph nodes, spleen, thymus, pituitary gland, thyroids (with parathyroids), adrenals, eyeballs, skeletal muscle (femur), bone (sternum, femur), skin (lower abdomen) and mammary gland. All retained tissues and organs were fixed in 10% neutralized buffered formalin, except for the testes and epididymides, which were fixed in Bouin’s solution. All tissues and organs to be examined microscopically were routinely processed for embedding in paraffin, sectioned and stained with hematoxylin and eosin. Those from the control and high-dose groups were all examined microscopically, as well as all gross lesions and the liver and kidney in the 5-, 25- and 100-mg/kg groups. The uterus and vagina of
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DOI: 10.3109/01480545.2013.851690
1 female in the 100-mg/kg/day group were also examined because a gross lesion was observed in the ovary. Statistical analysis Data for body weights, food intakes, hematological and blood chemical parameters, urine volume, urine osmotic pressure, organ weights, grip strength and locomotor activity counts were analyzed using Bartlett’s test for homogeneity of variance. If the variance was homogeneous at a significance level of 5%, one-way analysis of variance was performed. If there was a significant difference in this analysis, the difference between the control group and each of the treatment groups was analyzed by Dunnett’s test (Dunnett et al., 1964). If the variance was not homogeneous, Kruskal– Wallis’ test (Kruskal & Wallis, 1952) was employed. If there was a significant difference in this analysis, the difference between the control group and each of the treatment groups was analyzed by Dunnett’s nonparametric test. Defecation and urination data were analyzed using Kruskal–Wallis’ test. If there was a significant difference, the difference between the control group and each of the treatment groups was analyzed by Dunnett’s nonparametric test. Concerning detailed clinical examination, reflex test, daily clinical observation, necropsy and histopathology data, the significance of a strictly increased incidence of response in a dose group, as compared to the control group, was evaluated with Fisher’s exact test. Graded histopathological data were evaluated by Mann–Whitney’s U test.
Results ETBE had no apparent effect on mortality in any of the rats, although 1 male in the 25-mg/kg group on day 37 of treatment and 2 males in the 400-mg/kg group on days 77 and 137 were found dead during the study. The dead animal in the 25-mg/kg group showed swelling of the submandibular region and died showing hemorrhage from the gingival. On gross pathological examination, discoloration of some organs and enlargement of the heart and adrenal glands were observed, but this was not considered to be an effect of ETBE. In the 400-mg/kg group, neither macroscopic nor histopathological examinations of the dead animals revealed any abnormalities. Because the deaths occurred immediately after dosing and there were no abnormalities before administration on the day of death, intubation error was suspected. Treatment-related clinical findings were decreased locomotor activity in males and females of the 100- and 400-mg/kg groups and decreased respiratory rate and incomplete eyelid opening in males and females of the 400-mg/kg group, but these signs mostly disappeared by week 1. Salivation was observed transiently after dosing in males of the 25-, 100- and 400-mg/kg groups and in females of the 400-mg/kg group. There were no ETBE-related alterations regarding detailed clinical observations, reflex test, grip strength, motor activity count, body weight (Figures 1 and 2), hematology (Table 1) and urinalysis. Food intakes were significantly increased in males of the 400-mg/kg group on day 124 of administration and in females
700 0 mg/ka/day 5 mg/kg/day 25 mg/kg/day 100 mg/kg/day 400 mg/kg/day
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Figure 1. Body-weight changes in male rats that received ETBE for 180 days orally.
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36
43 50 57 64 Administration days
71
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92
120
148
176
Figure 2. Body-weight changes in female rats that received ETBE for 180 days orally. Table 1. Hematological examinations in rats received ETBE for 180 days orally. Dose (mg/kg/day)
0
5
25
100
400
Males Number of animals examined RBC (104/mL) WBC (102/mL) Hb (g/dL) Ht (%) MCV (FL) MCH (pg) MCHC (g/dL) Platelet (104/mL) Reticulocyte (%) PT (sec) APTT (sec)
10 894 47 98 17 15.4 0.4 45.3 1.4 50.7 1.8 17.3 0.6 34.1 0.4 110.7 8.5 1.5 0.3 16.2 1.7 25.8 2.7
10 912 26 85 17 15.8 0.3 46.8 1.1 51.3 1.9 17.4 0.7 33.9 0.3 108.0 11.8 1.4 0.3 16.2 1.3 26.9 2.3
10 888 32 94 23 15.4 0.5 45.4 1.6 51.2 1.6 17.4 0.5 34.0 0.4 109.8 8.7 1.5 0.3 16.6 1.5 25.0 2.6
10 890 31 88 26 15.6 0.5 46.0 1.5 51.7 1.7 17.6 0.4 33.9 0.3 107.2 9.9 1.4 0.4 17.1 1.4 28.0 3.0
10 901 40 114 28 15.5 0.5 45.7 1.2 50.8 1.5 17.2 0.5 33.8 0.6 114.6 9.5 1.4 0.3 16.2 1.4 24.5 3.5
Females Number of animals examined RBC (104/mL) WBC (102/mL) Hb (g/dL) Ht (%) MCV (FL) MCH (pg) MCHC (g/dL) Platelet (104/mL) Reticulocyte (%) PT (sec) APTT (sec)
10 799 38 54 10 14.8 0.6 44.0 1.8 55.1 1.3 18.6 0.4 33.7 0.3 95.8 7.9 1.5 0.2 14.2 1.0 19.8 2.1
10 806 39 81 20a 15.1 0.6 44.6 1.9 55.4 1.8 18.7 0.6 33.8 0.3 106.0 7.1a 1.7 0.4 14.6 0.6 20.8 1.6
10 792 29 78 24a 14.8 0.6 43.9 1.7 55.4 1.2 18.7 0.5 33.7 0.3 100.3 11.8 1.8 0.4 14.1 0.7 21.2 3.0
10 794 34 71 20 14.9 0.5 43.9 1.4 55.3 1.4 18.7 0.5 33.9 0.3 94.1 8.0 1.8 0.2 13.8 0.5 22.8 2.3
10 805 32 70 18 15.2 0.5 44.4 1.5 55.2 1.2 18.8 0.4 34.1 0.3b 96.4 6.4 1.6 0.4 14.0 0.7 21.3 1.5
Each value represents the mean SD. Significantly different from the control (p50.05). b Significantly different from the control (p50.01). a
of the 100-mg/kg group on day 180 and the 400-mg/kg group on days 96, 124 and 180. These changes were considered to be not treatment related because they were observed transiently without abnormal urine, fecal output and body-weight changes. Among the evaluated clinical chemistry parameters, a statistically significant increase in level of total cholesterol (153%), compared to the control, was noted in males of the
400-mg/kg group. No dose-related changes were noted for other parameters in males or for all parameters in females (Table 2). A significant increase in relative mean liver weight was noted in males and females of the 400-mg/kg groups (117 and 112% for males and females, respectively), compared to the controls. In male and female rats, relative kidney weights were increased in the 100- and 400-mg/kg groups, compared
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Table 2. Blood chemical examinations in rats received ETBE for 180 days orally. Dose (mg/kg/day) Males Number of animals examined AST (IU/L) ALT (IU/L) ALP (IU/L) ChE (IU/L) g-GGT (IU/L) T-Cho (mg/dL) TG (mg/dL) Glucose (mg/dL) T-Protein (g/dL) Albumin (g/dL) A/G ratio BUN (mg/dL) Creatinine (mg/dL) T-Bil (mg/dL) Ca (mg/dL) IP (mg/dL) Na (mEq/L) K (mEq/L) Cl (mEq/L) Females Number of animals examined AST (IU/L) ALT (IU/L) ALP (IU/L) ChE (IU/L) g-GGT (IU/L) T-Cho (mg/dL) TG (mg/dL) Glucose (mg/dL) T-Protein (g/dL) Albumin (g/dL) A/G ratio BUN (mg/dL) Creatinine (mg/dL) T-Bil (mg/dL) Ca (mg/dL) IP (mg/dL) Na (mEq/L) K (mEq/L) Cl (mEq/L)
0
5
25
100
400
10 75 19 31 6 147 29 56 14 0.4 0.2 58 18 64 24 150 14 6.1 0.3 2.9 0.2 0.89 0.08 12.8 1.4 0.29 0.05 0.07 0.01 9.5 0.3 5.2 0.6 143 1 4.3 0.2 104.8 1.1
10 87 24 34 13 150 26 49 15 0.5 0.2 55 9 62 14 137 14 6.1 0.2 2.9 0.1 0.90 0.07 14.3 1.5 0.29 0.05 0.07 0.02 9.5 0.3 5.4 0.5 143 1 4.3 0.2 105.1 1.2
10 89 30 46 18 164 46 73 26 0.6 0.4 70 16 80 24 150 18 6.1 0.3 2.8 0.2 0.87 0.08 12.9 1.8 0.26 0.03 0.07 0.03 9.5 0.4 5.3 0.5 143 1 4.4 0.2 104.9 1.6
10 90 24 35 12 136 24 63 21 0.5 0.2 65 21 67 41 153 15 6.2 0.3 3.0 0.1 0.93 0.08 13.3 1.6 0.28 0.05 0.06 0.02 9.5 0.4 5.2 0.7 143 1 4.3 0.3 104.8 1.7
10 92 38 42 18 186 63 79 22 0.8 0.5 89 18b 72 25 148 10 6.3 0.2 2.9 0.1 0.83 0.04 13.8 1.8 0.29 0.05 0.09 0.03 9.6 0.2 5.1 0.5 143 1 4.5 0.4 103.6 0.7
10 77 24 28 11 67 29 627 113 0.5 0.3 87 21 36 19 138 11 6.9 0.5 3.6 0.4 1.11 0.10 13.4 2.0 0.32 0.03 0.08 0.02 9.9 0.3 5.5 0.6 142 1 4.0 0.3 106.3 2.1
10 85 27 31 13 71 20 587 133 0.7 0.4 81 19 44 27 132 9 6.8 0.3 3.5 0.2 1.08 0.12 12.7 2.2 0.26 0.05 0.10 0.02 9.9 0.3 5.6 0.6 142 1 4.0 0.2 106.0 2.0
10 87 23 34 13 53 15 671 117 0.6 0.2 81 10 42 18 139 15 7.0 0.3 3.9 0.2 1.21 0.04 12.4 2.1 0.28 0.06 0.09 0.01 10.1 0.4 5.3 0.6 141 2 4.1 0.3 105.2 1.5
10 92 29 41 22 55 11 615 151 0.5 0.3 85 19 49 25 140 12 7.0 0.3 3.7 0.2 1.13 0.08 13.2 1.7 0.27 0.03 0.07 0.02 10.0 0.4 5.3 0.4 142 2 4.1 0.4 105.3 2.3
10 80 22 34 13 54 14 597 190 0.4 0.2 90 16 42 23 143 22 7.0 0.4 3.8 0.3 1.16 0.11 13.9 2.6 0.27 0.05 0.08 0.02 9.9 0.4 5.3 0.4 142 1 3.8 0.3 105.3 1.5
Each value represents the mean SD. Significantly different from the control (p50.01).
b
to the controls (112 and 121% respectively, for males; 111 and 115%, respectively, for females). Other organ weights were not altered by ETBE administration (Tables 3 and 4). Livers of males and females and kidneys of males were the only tissues with significant microscopic findings after ETBE administration. Hepatocytes of rats treated with 400 mg/kg/day of ETBE showed hypertrophy, characterized by enlargement of hepatocytes in the centrilobular area with homogeneously eosinophilic cytoplasm (Table 5; Figures 3 and 4). An increased incidence of hyaline droplets was observed in kidney of males of the 100- and 400-mg/kg groups, compared to the control (Table 5). Hyaline droplets were not observed in female rats at the same dose levels. a2uglobulin immunoreactivity was present in hyaline droplets of the renal proximal tubule epithelium of male rat kidneys.
Discussion Hypertrophy of the centrilobular hepatocytes with increased relative liver weights in males and females of the
400-mg/kg group are in line with findings after ETBE exposure at 500 mg/kg/day and above in a two-generation study in rats (Gaoua et al., 2004) and at 1750 ppm and above in inhalation exposure studies in rats and mice (Medinsky et al., 1999). Although liver cytochrome P450s (CYPs) were not measured in the present study, hypertrophy of the centrilobular hepatocytes and increased relative liver weights were considered to be related to induced metabolic enzyme activity because there were no hepatocellular necroses, and CYP2A6 is highly responsible for metabolism of ETBE in rats and humans (McGregor, 2007). An increase in the level of total cholesterol was noted in the 400-mg/kg male group. A relationship between cholesterol levels and induction of CYPs has been reported (Thomas, 1984; Ourlin et al., 2002; Kiyosawa et al., 2004; Oliveir et al., 2000); however, blood cholesterol was not affected in females in this study and in the 28-day repeated dose oral study dosed at 1000 mg/kg/day (Miyata et al., 2004). Therefore, the reason for the elevation of cholesterol is not clear.
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Table 3. Absolute organ weights in rats received ETBE for 180 days orally. Dose (mg/kg/day)
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5
25
100
400
Males Number of animals examined Liver (g) Kidney (g) Testis (g) Brain (g) Adrenal (mg) Body weight (g)
15 15.09 1.98 3.27 0.34 3.44 0.30 2.13 0.10 55.3 7.7 632.9 72.8
15 14.80 2.13 3.29 0.30 3.12 0.68 2.12 0.08 53.7 8.6 594.0 59.1
14 16.18 2.82 3.47 0.32 3.43 0.26 2.15 0.08 52.7 9.7 634.3 78.0
15 15.64 4.64 3.42 0.48 3.38 0.28 2.16 0.10 55.2 7.0 597.7 105.1
13 18.00 3.51 4.09 0.86b 3.43 0.28 2.22 0.07a 60.9 11.6 644.5 103.6
Females Number of animals examined Liver (g) Kidney (g) Ovary (mg) Brain (g) Adrenal (mg) Body weight (g)
15 8.08 1.10 1.88 0.20 70.0 18.7 1.98 0.06 68.4 12.1 347.2 40.4
15 7.75 0.95 1.89 0.16 71.0 21.7 1.98 0.08 66.8 10.4 329.1 47.1
15 8.03 0.71 1.88 0.15 73.8 16.6 1.98 0.06 69.5 15.3 340.1 30.7
15 8.27 1.17 2.02 0.21 67.7 17.7 1.97 0.10 68.0 7.8 339.0 35.7
15 8.83 1.13 2.07 0.23a 76.6 18.2 1.97 0.06 72.9 9.7 337.1 37.0
Each value represents the mean SD. Significantly different from the control (p50.05). b Significantly different from the control (p50.01). a
Table 4. Relative organ weights in rats received ETBE for 180 days orally. Dose (mg/kg/day)
0
5
25
100
400
Males Number of animals examined Liver (g/100 g) Kidney (g/100 g) Testis (g/100 g) Brain (g/100 g) Adrenal (mg/100 g) Body weight (g)
15 2.38 0.15 0.52 0.04 0.55 0.08 0.34 0.04 8.8 1.4 632.9 72.8
15 2.49 0.21 0.56 0.05 0.53 0.12 0.36 0.04 9.1 1.3 594.0 59.1
14 2.55 0.22 0.55 0.04 0.55 0.08 0.34 0.05 8.3 1.1 634.3 78.0
15 2.59 0.37 0.58 0.07b 0.58 0.09 0.37 0.06 9.4 1.3 597.7 105.1
13 2.79 0.19b 0.63 0.07b 0.54 0.06 0.35 0.05 9.6 2.1 644.5 103.6
Females Number of animals examined Liver (g/100 g) Kidney (g/100 g) Ovary (mg/100 g) Brain (g/100 g) Adrenal (mg/100 g) Body weight (g)
15 2.34 0.28 0.54 0.06 20.4 5.4 0.58 0.07 19.8 3.2 347.2 40.4
15 2.37 0.22 0.58 0.07 21.4 5.0 0.61 0.08 20.5 3.7 329.1 47.1
15 2.36 0.14 0.56 0.04 21.8 4.8 0.59 0.05 20.4 3.6 340.1 30.7
15 2.44 0.25 0.60 0.06a 20.0 4.9 0.59 0.07 20.2 2.8 339.0 35.7
15 2.62 0.18b 0.62 0.06b 22.8 5.5 0.59 0.07 21.7 2.6 337.1 37.0
Each value represents the mean SD. Significantly different from the control (p50.05). b Significantly different from the control (p50.01). a
Table 5. Histopathological findings in rats received ETBE for 180 days orally. Dose (mg/kg/day) Males Number of animals examined Liver Centrilobular hypertrophy of hepatocytes Centrilobular lipid droplets in hepatocytes Centrilobular necrosis of hepatocytes Focal necrosis of hepatocytes Microgranuloma Midzonal lipid droplets in hepatocytes Periportal lipid droplets in hepatocytes Heart Focal myocarditis Kidney Basophilic tubules
Grade
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15
15
15
15
þ þþ þ þ þþ þþ þ þþ þ þþ
0 0 0 1 0 0 1 0 3 1
0 0 0 0 1 0 0 0 1 0
0 1 1 1 1 1 0 0 3 2
0 0 0 1 0 0 1 1 4 0
6b 0 0 0 2 0 0 0 1 0
þ
7
NE
1
NE
5
þ þþ
1 0
0 0
2 0
3 0
5 1a (continued )
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Table 5. Continued
Dose (mg/kg/day) Degeneration of tubular epithelium Hyaline cast Increased hyaline droplets Mineralization in papilla Pelvic dilatation Pyelitis Solitary cyst in medulla Testis Diffuse atrophy of seminiferous tubules Inhibited spermiation and deep retention of spermatids Multinucleated giant cell formation Bone marrow No abnormalities detected Females Number of animals examined Liver Centrilobular hypertrophy of hepatocytes Hepatodiaphragmatic nodule Microgranuloma Heart Focal myocalditis Kidney Mineralization in corticomedullary junction Mineralization in pelvis Solitary cyst in medulla Bone marrow No abnormalities detected
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þ þ þ þþ þ þ þ þþ þ
0 0 0 0 0 0 4 0 1
0 0 0 0 0 0 4 0 0
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NE NE NE NE NE
NE NE NE NE NE
NE NE NE NE NE
3 1 1 1 1
15
NE
NE
NE
15
þ
þ, slight; þþ, moderate; NE, not examined. Significantly different from the control (p50.05). b Significantly different from the control (p50.01). a
Figure 3. Liver of a male rat treated with 400 mg/kg/day of ETBE orally (normal, 360).
Figure 4. Liver of a male rat treated with 400 mg/kg/day of ETBE orally. Centrilobular hypertrophy of hepatocytes (360).
It has been reported that kidney toxicity resulting from ETBE treatment in rats is associated with male rat-specific a2u-globulin-nephropathy (Medinsky et al., 1999; Gaoua, 2004). The same changes were also observed in male rats exposed to methyl tertiary-butyl ether (MTBE) (Leavens et al., 2009; Prescott-Mathews et al., 1997). In the present study, whereas kidney weights of both males and females were increased, only males exhibited increased hyaline droplets in kidney. The presence of a2u-globulin was
proven by an immunohistological method, confirming that these changes were the result of a2u-globulin nephritis. In females, histopathological changes of kidneys were not noted. Therefore, the kidney changes observed in males were considered to be rat specific and to be of less toxicological importance. Ataxia was observed in the 13-week inhalation study of ETBE (Medinsky et al., 1999) and MTBE (Dekant et al., 2001). However, neurotoxicity was not apparent on repeated
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inhalation treatment with ETBE (Dorman et al., 1997; Lington et al., 1997). In the present study of oral treatment for 180 days, decreased locomotor movement was observed in the 100-mg/kg and above groups, and oligopnea and incomplete eyelid opening in the 400-mg/kg group; however, these were judged not to be severe changes because they were transient and did not worsen with repeated administration. The transient salivation observed in all ETBE-treated males and females of the 400-mg/kg group was likely to be merely a reflex to a bitter taste of ETBE with negligible toxicological significance, because no such sign was induced with the inhalation route (Dorman et al., 1997; Medinsky et al., 1999). From the results of the present experiment, it was concluded that ETBE did not show severe toxicity and the NOAEL of ETBE for systemic toxicity was 100 mg/kg b.w./ day, because increased relative liver weights and hypertrophy of centrilobular hepatocytes were observed in males and females receiving 400 mg/kg b.w./day.
Acknowledgements The authors are grateful to Ms. Yuki Kimura and Ms. Yukie Ito for their technical assistance during the performance of this study.
Declaration of interest This study was supported by grants from the Ministry of Economy, Trade and Industry, Japan.
References Act on the Evaluation of Chemical Substances and Regulation of Their Manufacture, etc., Act No. 117. The current chemicals and dangerous substances regulation law in Japan; 1973. Dekant W, Bernauer U, Rosner E, et al. (2001). Biotransformation of MTBE, ETBE, and TAME after inhalation or ingestion in rats and humans. Res Rep Health Eff Inst 29–71; discussion, 95–109. Dorman DC, Struve MF, Wong BA, et al. (1997). Neurotoxicological evaluation of ethyl tertiary-butyl ether following subchronic (90-day) inhalation in the Fischer 344 rat. J Appl Toxicol 17:235–242. Dunnett CW. (1964). New tables for multiple comparison with a control. Biometrics 20:482–491.
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European Fuel Oxygenates Association (EFOA). (2006). ETBE technical product bulletin. Available from: www.efoa.eu/en/document/etbeproduct-bulletin- - -june-2006.aspx. Accessed on November 1, 2013. Fujii S, Yabe K, Furukawa M, et al. (2010). A one-generation reproductive toxicity study of ethyl tertiary butyl ether in rats. Reprod Toxicol 30:414–421. Gaoua W. (2004). Ethyl tertiary butyl ether (ETBE): two-generation study (reproduction and fertility effects) by the oral route (gavage) in rats. CIT Study No. 24859 RSR. Available from: http://cfpub.epa.gov/ ncea/cfm/recordisplay.cfm?deid¼199331. Accessed on April 10, 2013. Kiyosawa N, Tanaka K, Hirao J, et al. (2004). Molecular mechanism investigation of phenobarbital-induced serum cholesterol elevation in rat livers by microarray analysis. Arch Toxicol 78:435–442. Kruskal WH, Wallis WA. (1952). Use of ranks in one-criterion variance analysis. J Am Stat Assoc 47:583–621. Leavens TL, Borghoff SJ. (2009). Physiologically based pharmacokinetic model of methyl tertiary butyl ether and tertiary butyl alcohol dosimetry in male rats based on binding to alpha 2u-globulin. Toxicol Sci 109:321–335. Lington AW, Dodd DE, Ridlon SA, et al. (1997). Evaluation of 13-week inhalation toxicity study on methyl t-butyl ether (MEBE) in Fischer344 rats. J Appl Toxicol 17:S37–S44. McGregor D. (2007). Ethyl tertiary-butyl ether: a toxicological review. Crit Rev Toxicol 37:287–312. Medinsky MA, Wolf DC, Cattley RC, et al. (1999). Effects of a thirteenweek inhalation exposure to ethyl tertiary butyl ether on Fischer-344 rats and CD-1 mice. Toxicol Sci 51:108–118. Miyata K, Koga T, Shinoda K, et al. (2004). Twenty-eight-day repeateddose oral toxicity study of ETBE in rats [in Japanese, unpublished]. MOE (Ministry of Environment), MHLW (Ministry of Health, Labour and Welfare), METI (Ministry of Economy, Trade and Industry) of Japan. (2003). Chemical substances control law. Olivier LM, Kovacs W, Masuda K, et al. (2000). Identification of peroxisomal targeting signals in cholesterol biosynthetic enzymes. AA-CoA thiolase, HMG-CoA symthase, MPPD and FPP synthase. J Lipid Res 41:1921–1935. Organization for Economic Corporation and Development (OECD). (1981). OECD guideline for testing of chemicals; Chronic Toxicity Studies (452). Paris: OECD. Ourlin JC, Handschin C, Kaufmann M, Meyer UA. (2002). A link between cholesterol levels and phenobarbital induction of cytochromes P450. Biochem Biophyes Res Commun 291:378–384. Prescott-Mathews JS, Wolf DC, Wong BA, Borghoff SJ. (1997). Methyl tert-butyl ether causes alpha 2u-globulin nephropathy and enhanced renal cell proliferation in male Fischer-344 rats. Toxicol Appl Pharmacol 143:301–314. Thomas KD. (1984). Plasma cholesterol levels in adult male and female rats after chronic treatment with phenobarbitone. Isr. J Med Sci 20: 240–241.
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RESEARCH ARTICLE
A subchronic (180-day) oral toxicity study of ethyl tertiary-butyl ether, a bioethanol, in rats Katsumi Miyata, Takayuki Koga, Sunao Aso, Satsuki Hoshuyama, Syozo Ajimi, and Kotaro Furukawa CERI Hita, Chemicals Evaluation and Research Institute, Hita, Japan
Abstract
Keywords
A subchronic (180-day) toxicity study was conducted to evaluate the effects of ethyl tertiarybutyl ether (ETBE), a biomass fuel, in male and female rats. ETBE was administered at dose levels of 0, 5, 25, 100 and 400 mg/kg/body weight (b.w.)/day by gavage. No treatment-related adverse effects were observed at 5, 25 or 100 mg/kg. Centrilobular hypertrophy of hepatocytes was observed in males and females and their relative liver weights were increased, suggesting enhanced metabolic activity. From these results, we concluded that the no observed adverse effect level of ETBE was 100 mg/kg b.w./day under the conditions tested.
Ethyl tertiary butyl ether (ETBE), oral, rat, subchronic toxicity
Introduction Ethyl tertiary-butyl ether (ETBE) is industrially produced from ethanol and isobutylene and commonly used as an oxygenated additive of gasoline. ETBE offers equal or greater air-quality benefits compared to ethanol while being technically and logistically less challenging. ETBE does not induce evaporation of gasoline, which is one of the causes of smog, and does not absorb moisture from the atmosphere (EFOA, 2006; Fujii et al., 2010). The Japanese government has developed the Kyoto Protocol Achievement Plan, in which biomass fuels are planned to be introduced at 500 000 kL of crude oil equivalent per year for transportation purposes. To achieve this goal, the Japanese oil industry decided to introduce 840 000 kL of bioETBE as up to 7% ETBE-blended gasoline for automobile fuel from 2010. The Japan Petroleum Energy Center (JPEC) and the Ministry of Economy, Trade and Industry, Japan (METI), have been leading the project to assess the health risks of ETBE before the registration of ETBE pursuant to Japan’s chemical substance control law (1973). A series of analytic and toxicological studies demonstrated that ETBE is a persistent chemical in terms of biodegradability, but ETBE is not very bioaccumulative and is neither mutagenic nor ecotoxic. However, ETBE induced an increase of liver weight and centrilobular hepatocyte hypertrophy in rats after a 28-day repeated oral toxicity study (Miyata et al., 2004). Based on these results, ETBE was designated as a type II monitoring chemical substance according to the Japanese act, which Address for correspondence: Katsumi Miyata, Hita Laboratory, Chemicals Evaluation and Research Institute, 822 Ishii-machi 3chome, Hita 877-0061, Oita, Japan. Fax: +81-973-23-9800. E-mail: [email protected]
History Received 11 April 2013 Revised 26 August 2013 Accepted 13 September 2013 Published online 19 November 2013
means that the substance is not likely to undergo chemical transformation through natural processes. Systemic inhalation toxicity of ETBE has been well characterized in subchronic (90-day) studies using rats and mice. ETBE caused transient ataxia in Fischer 344 rats at a dose level of 5000 ppm (Dorman et al., 1997). Transient ataxia was also found in another subchronic inhalation study (Medinsky et al., 1999), in which both Fischer 344 rats and CD-1 mice exhibited the abnormality at 5000 ppm. Other major toxic findings observed in rats of the latter study were increased growth rate and slightly increased heart weights in females, degenerations of testes in males, and increased kidney and liver weights in both sexes. Mice have shown ataxia and liver abnormalities, such as increased weight, increased rate of cell turnover and/or centrilobular hypertrophy in males and females (Medinsky et al., 1999). Based on these results, the no observed adverse effect level (NOAEL) of ETBE for systemic toxicity was estimated to be 500 ppm in both rats and mice. However, long-term oral toxicity studies using rats and mice have hitherto not been conducted. The present study aimed at examining the potential longterm systemic toxicity of ETBE administered by the oral route for precise risk assessment, following Organization for Economic Cooperation and Development (OECD) test guideline 452 (OECD, 1981) chronic toxicity studies, in principle, with an exposure period of 180 days.
Methods Materials ETBE (CAS no.: 637-92-3) was supplied by Tokyo Chemical Industry Co. Ltd. (lot no. R74EE, 99% in purity; Tokyo,
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Japan). Stability of ETBE was verified by infrared spectroscopy before and after the dosing period, and purity was confirmed by gas chromatography analysis. Animals Specific pathogen-free Crl:CD (Sprague-Dawley) rats were used in this study. Eighty animals of each sex were purchased from Hino Breeding Center, Charles River Laboratories Japan, Inc. (Yokohama, Japan) at 4 weeks of age. After a 6-day quarantine and acclimatization period, 75 healthy animals of both sexes were selected. Animals were weighed and assigned to five groups in such a way as to equalize group means and standard deviations (SDs) of body weights. Each group consisted of 15 males and 15 females. Animals were 5 weeks of age at start of dosing and maintained in a barrier-sustained animal room with controlled temperature (21–25 C), humidity (40–70%), ventilation (10–15 times per hour) and lighting cycle (12 hours of artificial light [7:00 a.m. to 7:00 p.m.] and 12 hours of darkness) and were given an autoclaved pellet diet (MF; Oriental Yeast Co., Ltd., Tokyo, Japan) and local tap water ad libitum. They were housed individually in metallic brackettype cages with wire mesh floors throughout the study. Test substance administration ETBE was dissolved in olive oil and orally administered to animals with a stomach tube once-daily at approximately the same time in the morning. The oral route is one of the most probable routes of human exposure to ETBE, because leakage of ETBE from a gasoline tank may contaminate underground water. Dosing volume was 5 mL/kg body weight (b.w.), and the volume for each animal was calculated based on the most recent body weight. Dosing solutions were analyzed for the concentration of ETBE and were used within 7 days after preparation. Stability of ETBE in dosing solutions during the dosing period was confirmed by chemical analysis (data not shown). Dose levels were selected based on the results of a 28-day repeated-dose oral toxicity study in rats (Miyata et al., 2004), in which Crl:CD (Sprague-Dawley) rats, 5/sex/group, received ETBE orally at dose levels of 0, 15, 25, 50, 100, 150, 400 and 1000 mg/kg/day for 28 days. This short-term study revealed decreased spontaneous movement at 100 mg/kg and above, along with increased weights of the liver at 400 mg/kg. Therefore, a dose level of 400 mg/kg b.w./day was selected for the highest dose for the definitive study, and the lower dose levels were set at 100, 25 and 5 mg/kg b.w./day. Control animals received the vehicle only in the same manner as for the test-substance-treated groups. Study design This study was conducted in compliance with the OECD Chronic Toxicity Study (OECD, 1981) guideline for test chemicals in principle, with some modifications, and complied with the principles for Good Laboratory Practice (OECD no. 26, November 1997). The study also followed the Act on Welfare and Management of Animals of the Ministry of the Environment, Japan (Act No. 105, October 1, 1973), and Basic Policies for the Conduct of Animal Experiments in
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Research Institutions under the Jurisdiction of the Ministry of Health, Labor and Welfare, Japan (June 1, 2006). Any clinical signs were recorded daily, and individual animal weights and food consumption were measured once-weekly for the first 13 weeks of administration and once-monthly thereafter. Detailed clinical observations were performed once-monthly. During the last month of administration, reflex, grip strength and locomotor activity were assessed. Hematology and serum biochemistry Blood samples were obtained on the next day of the last treatment. Animals were fasted overnight, and blood samples were collected from the abdominal aorta of each 10 animals of a group under deep ether anesthesia. Hematological parameters were assessed on red blood cell (RBC) count, white blood cell (WBC) count, hemoglobin (Hb) concentration, hematocrit (Ht) value, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, reticulocyte count, prothrombin time (PT), activated partial thromboplastin time (APTT) and differential leukocyte count. Clinical chemistry parameters determined in serum samples were glutamic-oxaloacetic transaminase (AST), glutamicpyruvic transaminase (ALT), alkaline phosphatase (ALP), cholinesterase (Che), gamma-glutamyl transpeptidase (GGT), total cholesterol (T-Cho), triglyceride (TG), glucose, total protein, albumin, blood urea nitrogen (BUN), creatinine, total bilirubin (T-Bil), calcium, inorganic phosphorus (IP), sodium, potassium and chlorine as well as the calculated albuminglobulin (A/G) ratio. Urinary parameters were assessed on urine volume, color, turbidity, specific gravity, pH, protein, glucose, occult blood and urinary sediment. Pathology After blood samples were collected, all animals, including 10 animals of the clinical examination, were sacrificed humanely and underwent complete necropsy, including external examination. Tissues and organs collected from males and females, as appropriate, consisted of gross lesions, the trachea, lungs, submandibular and sublingual glands, esophagus, stomach, intestine (duodenum, jejunum, ileum, cecum, colon and rectum), pancreas, liver, heart, aorta, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicles, ovaries, uterus, vagina, brain (cerebrum, cerebellum and medulla/ pons), spinal cord (cervical, thoracic and lumbar), sciatic nerve, bone marrow (sternum and femur), axillar and mesenteric lymph nodes, spleen, thymus, pituitary gland, thyroids (with parathyroids), adrenals, eyeballs, skeletal muscle (femur), bone (sternum, femur), skin (lower abdomen) and mammary gland. All retained tissues and organs were fixed in 10% neutralized buffered formalin, except for the testes and epididymides, which were fixed in Bouin’s solution. All tissues and organs to be examined microscopically were routinely processed for embedding in paraffin, sectioned and stained with hematoxylin and eosin. Those from the control and high-dose groups were all examined microscopically, as well as all gross lesions and the liver and kidney in the 5-, 25- and 100-mg/kg groups. The uterus and vagina of
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1 female in the 100-mg/kg/day group were also examined because a gross lesion was observed in the ovary. Statistical analysis Data for body weights, food intakes, hematological and blood chemical parameters, urine volume, urine osmotic pressure, organ weights, grip strength and locomotor activity counts were analyzed using Bartlett’s test for homogeneity of variance. If the variance was homogeneous at a significance level of 5%, one-way analysis of variance was performed. If there was a significant difference in this analysis, the difference between the control group and each of the treatment groups was analyzed by Dunnett’s test (Dunnett et al., 1964). If the variance was not homogeneous, Kruskal– Wallis’ test (Kruskal & Wallis, 1952) was employed. If there was a significant difference in this analysis, the difference between the control group and each of the treatment groups was analyzed by Dunnett’s nonparametric test. Defecation and urination data were analyzed using Kruskal–Wallis’ test. If there was a significant difference, the difference between the control group and each of the treatment groups was analyzed by Dunnett’s nonparametric test. Concerning detailed clinical examination, reflex test, daily clinical observation, necropsy and histopathology data, the significance of a strictly increased incidence of response in a dose group, as compared to the control group, was evaluated with Fisher’s exact test. Graded histopathological data were evaluated by Mann–Whitney’s U test.
Results ETBE had no apparent effect on mortality in any of the rats, although 1 male in the 25-mg/kg group on day 37 of treatment and 2 males in the 400-mg/kg group on days 77 and 137 were found dead during the study. The dead animal in the 25-mg/kg group showed swelling of the submandibular region and died showing hemorrhage from the gingival. On gross pathological examination, discoloration of some organs and enlargement of the heart and adrenal glands were observed, but this was not considered to be an effect of ETBE. In the 400-mg/kg group, neither macroscopic nor histopathological examinations of the dead animals revealed any abnormalities. Because the deaths occurred immediately after dosing and there were no abnormalities before administration on the day of death, intubation error was suspected. Treatment-related clinical findings were decreased locomotor activity in males and females of the 100- and 400-mg/kg groups and decreased respiratory rate and incomplete eyelid opening in males and females of the 400-mg/kg group, but these signs mostly disappeared by week 1. Salivation was observed transiently after dosing in males of the 25-, 100- and 400-mg/kg groups and in females of the 400-mg/kg group. There were no ETBE-related alterations regarding detailed clinical observations, reflex test, grip strength, motor activity count, body weight (Figures 1 and 2), hematology (Table 1) and urinalysis. Food intakes were significantly increased in males of the 400-mg/kg group on day 124 of administration and in females
700 0 mg/ka/day 5 mg/kg/day 25 mg/kg/day 100 mg/kg/day 400 mg/kg/day
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Figure 1. Body-weight changes in male rats that received ETBE for 180 days orally.
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22
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36
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71
78
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92
120
148
176
Figure 2. Body-weight changes in female rats that received ETBE for 180 days orally. Table 1. Hematological examinations in rats received ETBE for 180 days orally. Dose (mg/kg/day)
0
5
25
100
400
Males Number of animals examined RBC (104/mL) WBC (102/mL) Hb (g/dL) Ht (%) MCV (FL) MCH (pg) MCHC (g/dL) Platelet (104/mL) Reticulocyte (%) PT (sec) APTT (sec)
10 894 47 98 17 15.4 0.4 45.3 1.4 50.7 1.8 17.3 0.6 34.1 0.4 110.7 8.5 1.5 0.3 16.2 1.7 25.8 2.7
10 912 26 85 17 15.8 0.3 46.8 1.1 51.3 1.9 17.4 0.7 33.9 0.3 108.0 11.8 1.4 0.3 16.2 1.3 26.9 2.3
10 888 32 94 23 15.4 0.5 45.4 1.6 51.2 1.6 17.4 0.5 34.0 0.4 109.8 8.7 1.5 0.3 16.6 1.5 25.0 2.6
10 890 31 88 26 15.6 0.5 46.0 1.5 51.7 1.7 17.6 0.4 33.9 0.3 107.2 9.9 1.4 0.4 17.1 1.4 28.0 3.0
10 901 40 114 28 15.5 0.5 45.7 1.2 50.8 1.5 17.2 0.5 33.8 0.6 114.6 9.5 1.4 0.3 16.2 1.4 24.5 3.5
Females Number of animals examined RBC (104/mL) WBC (102/mL) Hb (g/dL) Ht (%) MCV (FL) MCH (pg) MCHC (g/dL) Platelet (104/mL) Reticulocyte (%) PT (sec) APTT (sec)
10 799 38 54 10 14.8 0.6 44.0 1.8 55.1 1.3 18.6 0.4 33.7 0.3 95.8 7.9 1.5 0.2 14.2 1.0 19.8 2.1
10 806 39 81 20a 15.1 0.6 44.6 1.9 55.4 1.8 18.7 0.6 33.8 0.3 106.0 7.1a 1.7 0.4 14.6 0.6 20.8 1.6
10 792 29 78 24a 14.8 0.6 43.9 1.7 55.4 1.2 18.7 0.5 33.7 0.3 100.3 11.8 1.8 0.4 14.1 0.7 21.2 3.0
10 794 34 71 20 14.9 0.5 43.9 1.4 55.3 1.4 18.7 0.5 33.9 0.3 94.1 8.0 1.8 0.2 13.8 0.5 22.8 2.3
10 805 32 70 18 15.2 0.5 44.4 1.5 55.2 1.2 18.8 0.4 34.1 0.3b 96.4 6.4 1.6 0.4 14.0 0.7 21.3 1.5
Each value represents the mean SD. Significantly different from the control (p50.05). b Significantly different from the control (p50.01). a
of the 100-mg/kg group on day 180 and the 400-mg/kg group on days 96, 124 and 180. These changes were considered to be not treatment related because they were observed transiently without abnormal urine, fecal output and body-weight changes. Among the evaluated clinical chemistry parameters, a statistically significant increase in level of total cholesterol (153%), compared to the control, was noted in males of the
400-mg/kg group. No dose-related changes were noted for other parameters in males or for all parameters in females (Table 2). A significant increase in relative mean liver weight was noted in males and females of the 400-mg/kg groups (117 and 112% for males and females, respectively), compared to the controls. In male and female rats, relative kidney weights were increased in the 100- and 400-mg/kg groups, compared
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Table 2. Blood chemical examinations in rats received ETBE for 180 days orally. Dose (mg/kg/day) Males Number of animals examined AST (IU/L) ALT (IU/L) ALP (IU/L) ChE (IU/L) g-GGT (IU/L) T-Cho (mg/dL) TG (mg/dL) Glucose (mg/dL) T-Protein (g/dL) Albumin (g/dL) A/G ratio BUN (mg/dL) Creatinine (mg/dL) T-Bil (mg/dL) Ca (mg/dL) IP (mg/dL) Na (mEq/L) K (mEq/L) Cl (mEq/L) Females Number of animals examined AST (IU/L) ALT (IU/L) ALP (IU/L) ChE (IU/L) g-GGT (IU/L) T-Cho (mg/dL) TG (mg/dL) Glucose (mg/dL) T-Protein (g/dL) Albumin (g/dL) A/G ratio BUN (mg/dL) Creatinine (mg/dL) T-Bil (mg/dL) Ca (mg/dL) IP (mg/dL) Na (mEq/L) K (mEq/L) Cl (mEq/L)
0
5
25
100
400
10 75 19 31 6 147 29 56 14 0.4 0.2 58 18 64 24 150 14 6.1 0.3 2.9 0.2 0.89 0.08 12.8 1.4 0.29 0.05 0.07 0.01 9.5 0.3 5.2 0.6 143 1 4.3 0.2 104.8 1.1
10 87 24 34 13 150 26 49 15 0.5 0.2 55 9 62 14 137 14 6.1 0.2 2.9 0.1 0.90 0.07 14.3 1.5 0.29 0.05 0.07 0.02 9.5 0.3 5.4 0.5 143 1 4.3 0.2 105.1 1.2
10 89 30 46 18 164 46 73 26 0.6 0.4 70 16 80 24 150 18 6.1 0.3 2.8 0.2 0.87 0.08 12.9 1.8 0.26 0.03 0.07 0.03 9.5 0.4 5.3 0.5 143 1 4.4 0.2 104.9 1.6
10 90 24 35 12 136 24 63 21 0.5 0.2 65 21 67 41 153 15 6.2 0.3 3.0 0.1 0.93 0.08 13.3 1.6 0.28 0.05 0.06 0.02 9.5 0.4 5.2 0.7 143 1 4.3 0.3 104.8 1.7
10 92 38 42 18 186 63 79 22 0.8 0.5 89 18b 72 25 148 10 6.3 0.2 2.9 0.1 0.83 0.04 13.8 1.8 0.29 0.05 0.09 0.03 9.6 0.2 5.1 0.5 143 1 4.5 0.4 103.6 0.7
10 77 24 28 11 67 29 627 113 0.5 0.3 87 21 36 19 138 11 6.9 0.5 3.6 0.4 1.11 0.10 13.4 2.0 0.32 0.03 0.08 0.02 9.9 0.3 5.5 0.6 142 1 4.0 0.3 106.3 2.1
10 85 27 31 13 71 20 587 133 0.7 0.4 81 19 44 27 132 9 6.8 0.3 3.5 0.2 1.08 0.12 12.7 2.2 0.26 0.05 0.10 0.02 9.9 0.3 5.6 0.6 142 1 4.0 0.2 106.0 2.0
10 87 23 34 13 53 15 671 117 0.6 0.2 81 10 42 18 139 15 7.0 0.3 3.9 0.2 1.21 0.04 12.4 2.1 0.28 0.06 0.09 0.01 10.1 0.4 5.3 0.6 141 2 4.1 0.3 105.2 1.5
10 92 29 41 22 55 11 615 151 0.5 0.3 85 19 49 25 140 12 7.0 0.3 3.7 0.2 1.13 0.08 13.2 1.7 0.27 0.03 0.07 0.02 10.0 0.4 5.3 0.4 142 2 4.1 0.4 105.3 2.3
10 80 22 34 13 54 14 597 190 0.4 0.2 90 16 42 23 143 22 7.0 0.4 3.8 0.3 1.16 0.11 13.9 2.6 0.27 0.05 0.08 0.02 9.9 0.4 5.3 0.4 142 1 3.8 0.3 105.3 1.5
Each value represents the mean SD. Significantly different from the control (p50.01).
b
to the controls (112 and 121% respectively, for males; 111 and 115%, respectively, for females). Other organ weights were not altered by ETBE administration (Tables 3 and 4). Livers of males and females and kidneys of males were the only tissues with significant microscopic findings after ETBE administration. Hepatocytes of rats treated with 400 mg/kg/day of ETBE showed hypertrophy, characterized by enlargement of hepatocytes in the centrilobular area with homogeneously eosinophilic cytoplasm (Table 5; Figures 3 and 4). An increased incidence of hyaline droplets was observed in kidney of males of the 100- and 400-mg/kg groups, compared to the control (Table 5). Hyaline droplets were not observed in female rats at the same dose levels. a2uglobulin immunoreactivity was present in hyaline droplets of the renal proximal tubule epithelium of male rat kidneys.
Discussion Hypertrophy of the centrilobular hepatocytes with increased relative liver weights in males and females of the
400-mg/kg group are in line with findings after ETBE exposure at 500 mg/kg/day and above in a two-generation study in rats (Gaoua et al., 2004) and at 1750 ppm and above in inhalation exposure studies in rats and mice (Medinsky et al., 1999). Although liver cytochrome P450s (CYPs) were not measured in the present study, hypertrophy of the centrilobular hepatocytes and increased relative liver weights were considered to be related to induced metabolic enzyme activity because there were no hepatocellular necroses, and CYP2A6 is highly responsible for metabolism of ETBE in rats and humans (McGregor, 2007). An increase in the level of total cholesterol was noted in the 400-mg/kg male group. A relationship between cholesterol levels and induction of CYPs has been reported (Thomas, 1984; Ourlin et al., 2002; Kiyosawa et al., 2004; Oliveir et al., 2000); however, blood cholesterol was not affected in females in this study and in the 28-day repeated dose oral study dosed at 1000 mg/kg/day (Miyata et al., 2004). Therefore, the reason for the elevation of cholesterol is not clear.
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Table 3. Absolute organ weights in rats received ETBE for 180 days orally. Dose (mg/kg/day)
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5
25
100
400
Males Number of animals examined Liver (g) Kidney (g) Testis (g) Brain (g) Adrenal (mg) Body weight (g)
15 15.09 1.98 3.27 0.34 3.44 0.30 2.13 0.10 55.3 7.7 632.9 72.8
15 14.80 2.13 3.29 0.30 3.12 0.68 2.12 0.08 53.7 8.6 594.0 59.1
14 16.18 2.82 3.47 0.32 3.43 0.26 2.15 0.08 52.7 9.7 634.3 78.0
15 15.64 4.64 3.42 0.48 3.38 0.28 2.16 0.10 55.2 7.0 597.7 105.1
13 18.00 3.51 4.09 0.86b 3.43 0.28 2.22 0.07a 60.9 11.6 644.5 103.6
Females Number of animals examined Liver (g) Kidney (g) Ovary (mg) Brain (g) Adrenal (mg) Body weight (g)
15 8.08 1.10 1.88 0.20 70.0 18.7 1.98 0.06 68.4 12.1 347.2 40.4
15 7.75 0.95 1.89 0.16 71.0 21.7 1.98 0.08 66.8 10.4 329.1 47.1
15 8.03 0.71 1.88 0.15 73.8 16.6 1.98 0.06 69.5 15.3 340.1 30.7
15 8.27 1.17 2.02 0.21 67.7 17.7 1.97 0.10 68.0 7.8 339.0 35.7
15 8.83 1.13 2.07 0.23a 76.6 18.2 1.97 0.06 72.9 9.7 337.1 37.0
Each value represents the mean SD. Significantly different from the control (p50.05). b Significantly different from the control (p50.01). a
Table 4. Relative organ weights in rats received ETBE for 180 days orally. Dose (mg/kg/day)
0
5
25
100
400
Males Number of animals examined Liver (g/100 g) Kidney (g/100 g) Testis (g/100 g) Brain (g/100 g) Adrenal (mg/100 g) Body weight (g)
15 2.38 0.15 0.52 0.04 0.55 0.08 0.34 0.04 8.8 1.4 632.9 72.8
15 2.49 0.21 0.56 0.05 0.53 0.12 0.36 0.04 9.1 1.3 594.0 59.1
14 2.55 0.22 0.55 0.04 0.55 0.08 0.34 0.05 8.3 1.1 634.3 78.0
15 2.59 0.37 0.58 0.07b 0.58 0.09 0.37 0.06 9.4 1.3 597.7 105.1
13 2.79 0.19b 0.63 0.07b 0.54 0.06 0.35 0.05 9.6 2.1 644.5 103.6
Females Number of animals examined Liver (g/100 g) Kidney (g/100 g) Ovary (mg/100 g) Brain (g/100 g) Adrenal (mg/100 g) Body weight (g)
15 2.34 0.28 0.54 0.06 20.4 5.4 0.58 0.07 19.8 3.2 347.2 40.4
15 2.37 0.22 0.58 0.07 21.4 5.0 0.61 0.08 20.5 3.7 329.1 47.1
15 2.36 0.14 0.56 0.04 21.8 4.8 0.59 0.05 20.4 3.6 340.1 30.7
15 2.44 0.25 0.60 0.06a 20.0 4.9 0.59 0.07 20.2 2.8 339.0 35.7
15 2.62 0.18b 0.62 0.06b 22.8 5.5 0.59 0.07 21.7 2.6 337.1 37.0
Each value represents the mean SD. Significantly different from the control (p50.05). b Significantly different from the control (p50.01). a
Table 5. Histopathological findings in rats received ETBE for 180 days orally. Dose (mg/kg/day) Males Number of animals examined Liver Centrilobular hypertrophy of hepatocytes Centrilobular lipid droplets in hepatocytes Centrilobular necrosis of hepatocytes Focal necrosis of hepatocytes Microgranuloma Midzonal lipid droplets in hepatocytes Periportal lipid droplets in hepatocytes Heart Focal myocarditis Kidney Basophilic tubules
Grade
0
5
25
100
400
15
15
15
15
15
þ þþ þ þ þþ þþ þ þþ þ þþ
0 0 0 1 0 0 1 0 3 1
0 0 0 0 1 0 0 0 1 0
0 1 1 1 1 1 0 0 3 2
0 0 0 1 0 0 1 1 4 0
6b 0 0 0 2 0 0 0 1 0
þ
7
NE
1
NE
5
þ þþ
1 0
0 0
2 0
3 0
5 1a (continued )
Subchronic toxicity study of ETBE in rats
DOI: 10.3109/01480545.2013.851690
309
Table 5. Continued
Dose (mg/kg/day) Degeneration of tubular epithelium Hyaline cast Increased hyaline droplets Mineralization in papilla Pelvic dilatation Pyelitis Solitary cyst in medulla Testis Diffuse atrophy of seminiferous tubules Inhibited spermiation and deep retention of spermatids Multinucleated giant cell formation Bone marrow No abnormalities detected Females Number of animals examined Liver Centrilobular hypertrophy of hepatocytes Hepatodiaphragmatic nodule Microgranuloma Heart Focal myocalditis Kidney Mineralization in corticomedullary junction Mineralization in pelvis Solitary cyst in medulla Bone marrow No abnormalities detected
Grade
0
5
25
100
400
þ þ þ þþ þ þ þ þþ þ
0 0 0 0 0 0 4 0 1
0 0 0 0 0 0 4 0 0
1 0 0 0 0 0 1 1 0
1 1 4 0 1 0 0 0 0
0 1 5 5b 0 1 0 0 0
þ þþ þþ
0 0 0
1 1 1
NE NE NE
NE NE NE
0 0 0
15
NE
NE
NE
15
15
15
15
15
15
þþ
0 0 0
0 1 0
0 0 1
0 0 0
6a 0 0
þ
1
NE
NE
NE
0
þ þþ þ þþ þ
3 0 0 1 0
NE NE NE NE NE
NE NE NE NE NE
NE NE NE NE NE
3 1 1 1 1
15
NE
NE
NE
15
þ
þ, slight; þþ, moderate; NE, not examined. Significantly different from the control (p50.05). b Significantly different from the control (p50.01). a
Figure 3. Liver of a male rat treated with 400 mg/kg/day of ETBE orally (normal, 360).
Figure 4. Liver of a male rat treated with 400 mg/kg/day of ETBE orally. Centrilobular hypertrophy of hepatocytes (360).
It has been reported that kidney toxicity resulting from ETBE treatment in rats is associated with male rat-specific a2u-globulin-nephropathy (Medinsky et al., 1999; Gaoua, 2004). The same changes were also observed in male rats exposed to methyl tertiary-butyl ether (MTBE) (Leavens et al., 2009; Prescott-Mathews et al., 1997). In the present study, whereas kidney weights of both males and females were increased, only males exhibited increased hyaline droplets in kidney. The presence of a2u-globulin was
proven by an immunohistological method, confirming that these changes were the result of a2u-globulin nephritis. In females, histopathological changes of kidneys were not noted. Therefore, the kidney changes observed in males were considered to be rat specific and to be of less toxicological importance. Ataxia was observed in the 13-week inhalation study of ETBE (Medinsky et al., 1999) and MTBE (Dekant et al., 2001). However, neurotoxicity was not apparent on repeated
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inhalation treatment with ETBE (Dorman et al., 1997; Lington et al., 1997). In the present study of oral treatment for 180 days, decreased locomotor movement was observed in the 100-mg/kg and above groups, and oligopnea and incomplete eyelid opening in the 400-mg/kg group; however, these were judged not to be severe changes because they were transient and did not worsen with repeated administration. The transient salivation observed in all ETBE-treated males and females of the 400-mg/kg group was likely to be merely a reflex to a bitter taste of ETBE with negligible toxicological significance, because no such sign was induced with the inhalation route (Dorman et al., 1997; Medinsky et al., 1999). From the results of the present experiment, it was concluded that ETBE did not show severe toxicity and the NOAEL of ETBE for systemic toxicity was 100 mg/kg b.w./ day, because increased relative liver weights and hypertrophy of centrilobular hepatocytes were observed in males and females receiving 400 mg/kg b.w./day.
Acknowledgements The authors are grateful to Ms. Yuki Kimura and Ms. Yukie Ito for their technical assistance during the performance of this study.
Declaration of interest This study was supported by grants from the Ministry of Economy, Trade and Industry, Japan.
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